结核分枝杆菌抗原Rv0585c人T细胞抗原表位鉴定及其免疫原性评价

Identification and evaluation of T cell epitopes of Rv0585c from Mycobacterium tuberculosis

目的研究结核分枝杆菌抗原Rv0585c的抗原表位及其免疫原性，为结核病特异免疫诊断技术和疫苗的研发提供基础。方法利用生物信息学TE．predict和IEDB人T细胞抗原表位预测软件进行结核分枝杆菌抗原Rv0585c的人T细胞抗原表位预测，根据预测结果，合成表位多肽，用ELISpot试验检测预测表位在临床结核病患者中的免疫反应性。分别采用高、低剂量（100μg/只和50μg/只）的Rv0585c抗原表位多肽、同时以高、低剂量（50μg/只和20μg/只）的Ag85B蛋白抗原为对照，对随机分组的BALB／c小鼠进行免疫。利用ELISA方法检测IFN-γ、IL-2、IL-4、IL-10的水平。结果经生物信息学技术预测到Rv0585c66个人T细胞抗原表位，选取合成了表位分布集中的抗原表位多肽9条。人群ELISpot试验筛选出3条阳性人T细胞表位多肽：P10110、P10112、P10117，用于肺结核检测的灵敏度分别为14．00％、12．00％和6．00％，特异度分别为100．00％、100．00％和97．96％；联合用于肺结核检测的灵敏度和特异度分别为22．00％和97．96％。动物免疫试验结果显示，P10110多肽高、低剂量刺激小鼠产生较高水平的IFN-γ、IL-2、IL-4和IL-10，P10112多肽高、低剂量刺激小鼠产生较高水平的IFN-γ、IL-2和IL-10，均高于阴性对照组，差异有统计学意义（P〈0．001）。结论Rv0585c蛋白及其T细胞表位具有较好的免疫原性及免疫反应性，能刺激机体产生较强烈的细胞免疫应答，具有潜在的结核病细胞免疫诊断和新型结核疫苗的应用价值。

Objective To investigate the human T cell epitopes of Mycobacterium （M.） tuberculosis Rv0585c protein antigen and their immunogenicity and provide evidence for the development of specific tuberculosis immune diagnostic techniques and tuberculosis vaccine. Methods We synthesized peptides from M. tuberculosis Rv0585c protein antigen predicted by TE-predict and IEDB human T cell epitope prediction tool. The cellular immunoreactivity of the predicted peptides was evaluated through ELISpot assay with the peripheral blood monouclear cells （PBMC） of clinical tuberculosis patients. In animal experiments, BALB/c mice were respectively immunized with high dose （100 gg/mice） and low dose （50 gg/mice） of the peptides of Rv0585c, at the same time, high dose （50 gg/mice） and low dose （20 gg/mice） of Ag85B protein were used in positive control group. The levels of IFN-γ, IL-2, IL-4 and IL-10 were tested with ELISA kit respectively. Results By means ofbioinformatics technique, 66 human T cell epitopes of Rv0585c were predicted, from which 9 peptides concentrated epitopes were synthesized for the animal immune experiments. Peptides P 10110, P 10112 and P 10117 were confirmed to be antigenic. The sensitivity and specificity of P10110, P10112 and P10117 were 14.00%, 12.00%, 6.00% and 100.00%, 100.00%, 97.96% respectively when they were used as diagnostic reagents of tuberculosis. The sensitivity and specificity were 22.00% and 97.96% when the epitopes were combined together. The results of animal immunity test showed that high levels of cytokines IFN-γ, IL-2, IL-4 and IL- 10 were induced by high and low dose of P10110, and high levels of IFN-γ, IL-2 and IL-10 were induced by high and low dose of P10112, which were much higher than that in negative controls, respectively （P〈0.001）. Conclusion Rv0585c, including its human T cell epitopes, has good immunogenicity and immunoreactivity, stimulating the body to produce a stronger cellular immune response and has better potential application value in cellular diagnosis of tuberculosis and the development of new type of tuberculosis vaccine.